Method of tissue repair II

ABSTRACT

A substantially solid biomolecular solder for joining tissue comprising a partially denatured biomolecule. The solder can be formed into shapes to suit the needs of a user. The invention also relates to methods for joining tissue and methods for preparing the solder.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application, filed under 35 U.S.C. 371, claims the benefit ofpriority to international application PCT/AU99/00495, filed Jun. 18,1999, which was published under PCT Article 21(2) in English, and ishereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to methods for joining: living tubulartissues; organs and their coverings; skin and appendages; as well as thevarious internal and peripheral nerves of the body, the spinal cord andits ramifications. The invention also relates to a solder for use inthose methods and methods for preparing the solder.

BACKGROUND ART

In repairing living tissues, sutures or clips are routinely used toclose defects, join planes of tissues or to join bodily tubes together(anastomoses).

This involves the placing of materials in the body which cause somedamage to the tissues involved, but hold those tissues in appositionwhile the body's own healing processes effect a more permanent join. Thedamage that various joining materials cause varies but even carefulplacement of microsutures in the smallest of bodily tubes during ananastomosis produces a fibrous tissue reaction around each of the suturematerials left in situ.

Joins, however made, take time, and those joins made by placingindividual sutures in tubular joins are the most time consuming. Sewingin a ring of sutures to effect such a join inside the body may demand alarge incision to obtain the access required to effect enough surgicalfreedom to manipulate the equipment and instruments required.Microsuturing requires considerable skill.

Arteries and Other Tubes

Fluids, and materials suspended within them, can travel along the body'spatent tubes. Arteries carry blood from the heart to other organs andtissues in the body. They have 3 layers, an inner specialised mucosa(termed the intima), a thicker, middle, muscular and structural layerwhich contains collagen and elastin connective proteins (the media), andan outside layer which is a scaffold with fibrous tissue, blood vesselsand nerves all supplying the functions of the artery (the adventitia).The inner volume of the artery is the lumen.

For tubes such as arteries to function in transporting blood at highpressure, they need to be strong. They are actually active intransporting a pressure wave of blood by expanding and relaxing (systoleand diastole) as the bolus of blood passes. Joining such active tubesrequires such physiological activity as promoting blood flow to beconsidered and the design of methods of anastomosis that will allow theactivity to continue after the join.

Injuries to an artery are potentially very serious for an animal orhuman, as blood flowing through the artery is at high pressure and bloodloss can be rapid. If the intima layer is damaged, then the middle,structural layer, the media, is exposed to blood. This triggers animportant repair mechanism which acts to seal the wound and preventfurther bleeding by the formation of blood clots on the wound, caused byblood coming into contact with the exposed collagen of the media.

Although microsuturing is the standard clinical repair technique for asevered artery, it has several disadvantages. A high skill level isrequired to make between 6 and 12 separate sutures to repair the artery.The sutures remain in the body acting as a site for fibrous tissue toform due to foreign body reaction, and this fibrous tissue is a point ofweakness in the artery even after it is deemed to have healed. Althoughsuturing does not produce a fluid-tight seal, surgeons usually rely onblood clotting triggered by the mechanism described above to seal thevessel soon after the repair is complete.

A number of laser-assisted welding techniques have been explored inorder to find a more convenient technique which does not lead to so muchscarring. These almost always need stay sutures (sutures used to jointhe vessels before laser treatment, which may or may not be removedsubsequently) for a successful outcome. In this case the two vessel endsare held together to allow stay sutures to be inserted and then a laseris used to heat the tissue at the join so that proteins at the site arecoagulated and bond together. Lasers such as the infra-red holmium-dopedYAG and carbon dioxide lasers have been used because these producewavelengths which are strongly absorbed by water in the tissue.Alternatively a dye solution may be applied to the tissue to enhancelight absorption at a suitable laser wavelength. In any case, it iscrucial that the intima layers of the 2 ends are in continuity, to avoida blockage or a clot and to promote smooth laminar flow in the repairedvessel. This is difficult to achieve in thick-walled vessels where thelaser energy may not be absorbed through all three layers of the vesselto form a strong weld with a smooth intima layer.

Some protein glues have been used to repair blood vessels, such asfibrin (which triggers a blood clotting reaction to effect a tissuejoin). A possible disadvantage of such a glue is the potential to beassociated with blood clotting within the vessel, partially or whollyobstructing it.

Laser-activated fluid albumin solder has also been used, but the solderhas required stay sutures to achieve sufficient repair strength forarteries which carry blood at high pressure. Fluid glues and solderstend to run between the tissue ends, risking blockage of the innerlumen, and are difficult to control and position accurately on thetissue repair. To attain a seal, they have been appliedcircumferentially around the join, which is then circumferentiallywelded. These joins later show thick scarring which can cause strictureor blockage of the vessel or tube.

There is also a lack of precision in such techniques, because ofdifferences in the glue or fluid solder consistency, variations in thetype of applicator device used to apply the glue or fluid solder, andthe pressure needed to form a join.

A major drawback with current fluid solders is that they rapidlydeteriorate and change composition when introduced into moistenvironments.

Similarly, existing solid solders must be kept dry when introduced tomoist arteries, to prevent them from absorbing moisture, weakening theirinternal bonding and losing strength, even though this occurs moreslowly than for fluid solders.

The repair of other bodily tubes is similar in is concept. Since thestructure of each tube is specialised to its function and the nature ofits contents there must be careful choice of the method of tube repairso that it will not interfere with the tube function, and in particularwith maintaining the inner lumen of the tube.

Peripheral Nerves

The electrical signals that control the body's organs and transmitinformation back and forth to the central nervous system (CNS) travelalong peripheral nerves.

A peripheral nerve has an outer membrane consisting of connective tissuesuch as collagen. This membrane (epineurium) protects and holds separatebundles of nerves or fascicles together. The fascicles group togethernerve axons supplying a specific region of the body and are bounded byperineurium membranes. Each axon is supported by a Schwann cell withinthe fascicle. Nerve metabolism is sustained by the vascular system fromboth outside and within the nerve.

When a peripheral nerve is cut all axons distal (further from the spine)to the wound change their properties. Even when the nerve isreconnected, these axons continue to degenerate distally. The Schwanncells which normally wrap themselves around the axons as insulation,guide regenerating axons. Joining nerves as accurately as possible bylining up corresponding fascicles enables the enclosed axons to moreefficiently regenerate.

Peripheral nerves can have diameters ranging from approximately 1 cm toapproximately 50 micrometers.

Operating on nerves and other tissues of small dimensions has beenfacilitated by using magnification and special microsurgical equipment.Accurate nerve repairs need to be effected at the fascicular levelensuring that regeneration is along the correct bundle leading to theoriginal area those axons supplied.

The current technique of peripheral nerve repair uses microsuturing.This technique requires a dedicated trained surgeon as microsuturing ofjust one of the many fascicles with three or more microsutures (usingsay a 70 micron diameter needle and 30 micron thread) can take very longoperating times. There is the prospect of added damage to the inneraxons due to sutures penetrating the thin perineurial sheath. The use ofsutures results in some scarring of the repair due to foreign bodyreaction. Excessive scarring impairs nerve function and may beassociated with painful neuromas. There is also evidence that in thelong term, scar tissue formation and scar maturation can impair thejoined nerve.

Work has been performed on the use of lasers alone in effecting nervejoins. To date the welds have typically been made using infrared laserssuch as carbon dioxide lasers which rely on water absorption forenergy-transfer. Tissue preparation before welding relies on overlappingthe nerve membranes. One of the problems of laser welding has been thefact that the intact axonal tissue is under pressure within thefascicle, so that when it is cut the axons extrude. Laser treatment canthus lead to denaturation of the axon material leading to scarring andproliferation of fibrous tissue.

Laser-activated protein solders have also been tried, as described forthe artery and blood vessel case above. Again because of difficulties incontrolling fluid solders, and the weakness of the resulting bonds in amoist environment, these repairs are usually too weak without theaddition of stay sutures. This complicates the surgical technique andleads to additional scarring and foreign body reaction.

The bonds formed to date as described in the prior art using laserwelding have typically lacked strength and thus microsuturing has beenused in addition to welding to strengthen these joins.

Solutions to at least some of these problems are taught in WO96/22054.The present invention relates to alternative solutions.

DESCRIPTION OF THE INVENTION

In a first aspect the present invention provides a biomolecular soldercomprising an at least substantially solid composition of at least onebiomolecule which has been mixed at high concentration with an aqueoussolvent, which composition is created to at least partially denature thebiomolecular components of the solder and to at least partly dry thesolder.

The biomolecule(s) is typically proteinaceous but it is envisaged thatother naturally occurring biomolecules could be used as alternatives.Further, analogues of biological, biodegradable polypeptides could beused. Analogues of biological, biodegradable polypeptides useful in thesolders of the invention include synthetic polypeptides and othermolecules capable of forming the solder of the invention but which donot cause adverse reaction in the tissue undergoing repair.

Where the biomolecule is a protein, the protein can be any protein ormixture of proteins but is preferably bio-degradable in the relevanthost. Examples of suitable proteins include albumins, collagen,fibrinogen and elastin. Suitable proteins are typically those which canbe cross-linked to form a matrix and which can be resorbed by the body.Where combinations of proteins are used it is envisaged that thosecombinations will be of proteins having similar denaturationtemperatures. An example is the combination of albumin and collagen. Useof different albumins is contemplated including bovine, horse, human,rat, ovine and rabbit albumin. The choice of a particular albumin may bemade to reduce immunological reaction in the patient to the solder. Itis envisaged that there will be circumstances where the albumin used maybe chosen to match the patient's blood type and possibly even morespecifically with regard to histocompatibility markers of the patient inquestion.

The solvent is typically water but other aqueous solvents includingsaline may be used provided that any salt etc present does not adverselyaffect the solder upon denaturation.

The solder can be formed from a protein paste made up of highlyconcentrated protein in an aqueous solvent which is typically water.Highly concentrated protein encompasses protein concentrations in therange of 40 to 80% w/w. Preferably the protein concentration is in therange of 45 to 75% w/w. More preferably, the protein concentration is inthe range of 50 to 60% w/w. The range of 50 to 60% is especiallypreferred for bovine serum albumin, or rat or rabbit or ovine or humanalbumin. The starting concentration of protein loses water (or aqueoussolvent) as it dries or is dried during processing. The prepared soldermay contain little or no solvent.

It is preferred to incorporate light-absorbing material, such as a dye,into the solder, to improve energy deposition in the solder. An exampleof a suitable dye is indocyanine green which is preferably incorporatedat a concentration within the range 0.1 to 2.5% w/w. Other suitable dyesinclude methylene blue and fluorescein isothiocyanate. It will beunderstood that the light-absorbing material is chosen to be appropriateto the energy source that is used in forming tissue repairs involvingthe use of the solder. The light absorbing substance may be incorporatedby being added to the solvent and dissolved in it prior to addition ofthe biomolecule(s) to the solvent.

In one embodiment the solder is prepared from a composition of:

55–75% w/w albumin

45–25% w/w water

0.25% w/w indocyanine green

The albumin may be bovine, rabbit, human, ovine or rat albumin.

The at least partial denaturation of the biomolecule(s) substantiallyreduces the solubility of the solder. Typically the biomolecule(s) ofthe solder is denatured to a sufficient extent to ensure that the solderwill have sufficient longevity in vivo for the repair, for which thesolder is being used, to be formed. Denaturation favourably alters themechanical properties of the solder so that on moistening it exhibitssimilar mechanical properties to the tissue under repair. Thedenaturation can be effected by heat, light, radiation, ultrasound orchemical means. Typically the heat denaturation is carried out in anaqueous environment such as in a water bath in steam or in pressurisedsteam. Without wishing to be bound by theory, the present inventorsbelieve that the aqueous environment permits at least partialdenaturation without dissolution and with the maintenance of“structural” water involved in the integrity of the biomolecule(s).Denaturation may be effected before, during or after shaping of thesolder.

The solder can be provided in a variety of shapes. In particular, thesolder of the invention is suitable for extruding into tubular forms, aform that cannot readily be achieved with prior art solders. It can alsobe extruded into a partial tube which has a curved cross section with anelongate open channel which can be wide or narrow. The solder can beprepared with a smooth surface or with a surface that is at leastslightly roughened. Roughening may be of assistance in enhancing contactbetween tissue and solder. The roughening may provide a profile whichappears smooth at macroscopic level but rough at microscopic level. Thetubular and partially is tubular forms typically have a round or ovoidprofile but other profiles are also contemplated including squarecrenulated and other geometric forms. The tubular solder of theinvention can be tapered or of uniform cross section. The tubular solderof the invention is well suited to nerve repair applications and isparticularly well suited to vascular applications in which the moisturecontent makes prior art solders unsuitable. The solder can be preparedin other shapes as required for particular applications includingstrips, patches, solid rods and hollow cubes with at least one flangedend.

Various adjuvants can be added to the solder to promote rapid or morecomplete tissue healing, eg fibrinogen (for blood vessels), growthfactors, sodium hyaluronate (for improved viscous handling and possiblybetter healing), hormones, and/or anticoagulants, such as heparin.

Various fibrous materials can be added to the solder to improve thestrength of the solder [eg collagen or polytetrafluoroethylene fibre(which is sold under the brand names goretex and teflon) or ceramicfibres]. The fibres are typically biocompatible polymers. Thedenaturation of the solder with fibrous materials within it may be bychemical means (such as with acid or hydroxide) or by heat and couldinclude bonding of the protein to the fibres.

The solder need not be of uniform composition throughout. In someapplications it will be desirable to include one or more adjuvants inone or more parts of the solder and not in others. Similarly, it may bedesirable to incorporate fibres in some parts and not others or elsedifferent fibres in different parts. Further, one or morelight-absorbing substances may be incorporated in some parts of thesolder and not others or the light-absorbing substance may beincorporated at different concentrations throughout one or more parts ofthe solder. It will be recognised that such variations may beparticularly useful with various shaped forms of the solder such astubes. Still further, different parts of the solder may be denatured todifferent extents and different parts of the solder may be provided withdifferent surface textures, such as being smooth in some parts and atleast slightly roughened in other parts.

The solder can be applied to a mesh, stiffener or graft material madefrom, for instance, a metal, synthetic fibre or plastic. Because of itspliability, the solder may be embedded into spaces in the mesh or it maybe applied as a covering to all or part of the mesh, stiffener or graftmaterial. In one embodiment, it may be applied only to the ends of agraft material, mesh or stiffener to effect welding of the graftmaterial, mesh or stiffener to the appropriate tissue.

The formation of such materials may involve coextrusion or coating of abiologically inert porous structure (such as a goretex tube or shape)with solder. Where a coating is utilised in this embodiment, the soldermay be initially formulated in a fluid form, that is, with asubstantially lower concentration of the biomolecule(s). The fluidsolution is applied, allowed to dry and may be reapplied and allowed todry before being at least partially denatured. The drying processreduces the solvent content so that the final consistency of the solderis the same as that achieved by forming the solder from the highconcentration solution as described above.

The solder of the invention can be introduced to the relevant tissue bythe surgeon, and placed in the correct position, using forceps. Ifnecessary, the solder can be cut to a required size or shape duringsurgery.

The at least partially denatured biomolecule(s) of the solder has stronginternal bonding and is substantially unaffected by water absorption.Any water absorption that occurs acts to enhance the flexibility of thesolder rather than causing its dissolution or disruption.

The solder can be introduced into the relevant tissue in anappropriately moistened form. In this form the solder is flexible andwill not fracture when cut, squeezed or manipulated with surgicalinstruments.

The solder can be sterilised after denaturing and before use, by forinstance gamma ray irradiation, for instance at 2000 rad/min for 50minutes. Other suitable forms of sterilisation include autoclaving,steam treatment and heat treatment.

Activation of tissue bonding by the solder is induced by heat. This canbe achieved in a variety of ways but laser activation is the mostcommon. Because the biomolecule(s) is already at least partiallydenatured, dissolution is at least substantially prevented, allowingtime for more complex manipulations to be completed. Laser activation ofbonding through overlying tissue is possible with this solder, that is,the solder can be applied under, over, or under and over the tissue tobe joined.

In a second aspect the present invention provides kits of solder tubes,partial cubes and shapes formed from solder of the first aspect of theinvention. The kits may comprise tubes, partial tubes and/or shapes ofdifferent sizes to suit different surgical applications. The differentsized tubes can include different lumen sizes, wall thicknesses andlengths. It is envisaged that tubes will often be cut to length to suitthe repair to be effected during surgery thus minimising the number ofdifferent lengths that need to be provided. The kits can include tubes,partial tubes or shapes fashioned from solders made with differentbiomolecules, including those made with biomolecules which reflect theneed to match the repair material for histocompatibility markers in theanimal or human patient in which the repair is to be made. Further thetubes, partial tubes or shapes can be provided in different versionsincluding a series of different adjuvants, light-absorbing substancesand/or fibres as well as with different solder compositions throughoutthe tubes, partial tubes or shapes.

In a third aspect the present intention provides a method of preparing asolder of the first aspect, the method comprising the steps of forming ahigh concentration solution of one or more biomolecule(s) in an aqueoussolvent, at least partially denaturing the biomolecule(s) and drying thesolder.

Typically the method includes forming the solid solder into a shapewhich is preferably a hollow tube. Other suitable shapes include partialtubes, strips, patches, hollow tubes with at least one flanged end orsolid rods suitable for the tissue being repaired.

To form hollow tubes, the solder can be extruded into hollow tubes bythe use of a high pressure extrusion and die set, manufactured ofstainless steel or other suitable biologically inert material, which mayhave very smooth surfaces to permit smooth solder shapes to be extruded.Shaped solders can also be prepared by injection moulding.Alternatively, the extruded solder may be prepared with an at leastslightly roughened surface to enhance contact between the solder and thetissue to which it is applied. In this form, the solder may have asurface which is roughened on a microscopic scale but appears smooth ona macroscopic scale. The tube dimensions can be in the range of 0.2 mmto 6 cm in diameter, with variable wall thickness, which depending onthe cube diameter and strength of the solder, can be as low as 50 μm. Itwill be understood that for veterinary applications, where very largeanimals and very small animals may be involved that even greaterdiversity of tube sizes may be required to suit the needs of variousphysiological tubes in need of repair. The solder of the intention issuited to the precision manufacture of tubes of desired dimensions.

In one embodiment, the method for forming a tubular solder comprisesforming a high concentration solution of at least one biomolecule in anaqueous solvent, extruding the solution without permitting it to dry,allowing the extruded material to dry, at least partially denaturing theextruded material, allowing the at least partially denatured, extrudedmaterial to dry, moistening the material, cutting the material tolength, finally drying the material and sterilising the material.

The starting concentration of biomolecule loses aqueous solvent as itdries or is dried during processing. In the prepared solder, little orno solvent may be present.

The method may include incorporating a light-absorbing material, such asa dye, into the solder, to improve light energy deposition in thesolder, with the light-absorbing material being chosen to be appropriateto the energy source that is used in forming tissue repairs involvingthe use of the solder. Where a light-absorbing material is incorporatedthis may be achieved by mixing the light absorbing substance into thesolvent and then adding this solution to the biomolecule(s) for mixing.

Indocyanine green dye (for example, prepared at a concentration of 0.25mg/ml where the solder is placed over the tissue to be joined and 2.5mg/ml where the solder is placed under the tissue to be joined) can beincorporated into albumin protein paste (approximate concentration onmixing 60% weight/weight) which is then preferably denatured by theimmersion of the protein solder in a water bath at elevated temperature(preferably around 85° C.) for a suitable period of time (preferably 30seconds) or in steam where temperatures over 10° ° C. are used.

Typically the biomolecule(s) of the solder is denatured to a sufficientextent to ensure that the solder will have sufficient longevity in vivofor the repair for which the solder is being used to be formed. Thedenaturation can be effected by physical leans such as beat (direct orindirect), light, radiation or ultrasound or chemical means. Typicallythe denaturation is carried out in an aqueous environment such as awater bath in steam or in pressurised steam. This can be achieved wherethe biomolecule(s) is proteinaceous by immersing the protein paste inhot liquid (preferably water) at a temperature of over 40° C.(preferably 85° C. for bovine se albumin (BSA)) for a suitable time(preferably 30 seconds for (BSA) or in steam where temperatures over100° C. are used, for a suitable period of time. For human or rabbitserum albumin, steam treatment by for instance autoclaving attemperatures between 100° C. and 150° C. are preferred, withtemperatures between 110° C. and 130° C. being more preferred example ofa suitable temperature is about 120° C. The steam treatment is typicallyfor about 10 minutes. Denaturation may be effected before, during orafter shaping of the solder.

The method can include the addition of various adjuvants to the solder,eg fibrinogen (for blood vessels), growth factors, sodium hyaluronate(for improved viscous handling and better healing), hormones, and/oranticoagulants, such as heparin.

The method can also include the incorporation of various fibrousmaterials into the solder co improve the strength of the solder [egcollagen or polytetrafluoroethylene fibre, or ceramic fibres]. Thefibres are typically biocompatible polymers. The denaturation of thesolder with fibrous materials within it may be by chemical means (suchas with acid or hydroxide) or by heat and could include bonding of thebiomolecule(s) to the fibres.

The method may be modified to produce a solder that is not of uniformcomposition throughout. For instance, in some applications it will bedesirable to include one or more adjuvants in one or more parts of thesolder and not in others. Similarly, it may be desirable to incorporatefibres in some parts and not others or else different fibres indifferent parts. Further, one or more light-absorbing substances may beincorporated in some parts of the solder and not others or thelight-absorbing substance may be incorporated at differentconcentrations throughout one or more parts of the solder. A gradient orprofile of the concentration of light-absorbing material can be providedwithin the solder to control the heat deposition within the solder andavoid excessive thermal tissue damage. The gradient can be createdduring the preparation of the solder or by painting on a dye solutionafter solder tube formation. Still further, different parts or thesolder may be denatured to different extents. Still further, the soldermay be prepared with part of the surface at least partly roughened andpart of the surface smooth.

The method may include sterilising the solder after denaturing andbefore use. Suitable means of sterilisation include gamma rayirradiation, for instance at 2000 rad/min for 50 minutes, autoclaving,steam treatment, heat treatment and gas sterilisation.

Final denaturation of the solder occurs in situ in the tissue, byapplication of laser or other energy source, where the energy isabsorbed by the solder and/or the tissue.

In a fourth aspect the present invention provides a method of repairinga biological tissue comprising the use of a solder of the first aspectin effecting the repair.

The method can be used for effecting repairs in animal as well as humanpatients.

Typically, the method involves the use of an energy source such as alaser for effecting tissue joins using the solder. Where the energysource is a laser, the selected laser has a wavelength appropriate toany light-absorbing substance used to concentrate the energy at therepair site. The laser chosen should also be appropriate to the tissuebeing repaired in that the tissue absorbs the energy produced by thelaser poorly. For blood vessels, the combination of diode lasers withindocyanine green dye is appropriate. The energy provided should besufficient to bond the solder to the underlying or overlying tissuewhile minimising damage to the underlying tissue. The power used willvary for different tissues and can be matched to the amount of energyoutput required to effect bonding.

The time of treatment for each bond to be effected can vary depending onsuch factors as ambient conditions, altitude, humidity and the nature ofthe tissue being joined as well as the moisture level of the tissuebeing joined.

In one embodiment the invention provides a method for joining body tubescombining the use of a tubular solder of the first aspect and a laserfusion device. The tubular solder can be applied (depending on thephysiological tube to be repaired) to either fit inside or outside orinside and outside both the cut ends of the tube. The lasering may bedone either directly or through the living tube to the solder to changeits characteristics to make it adhesive.

The solder tube can incorporate a light-absorbing material to absorb thewavelength of the laser beam which is applied to form the bond.

Bonding can involve attaching at least one edge of the circumference ofthe solder tube to the inside or outside of the cylindrical surface of abody tube. The join of the body tube can be completed by placing bothends of the tube within the solder tube and applying energy through thesolder to bond the solder to the underlying tissue or by placing bothends of the body tube over the solder tube (FIG. 10) and applying energythrough the overlying tissue to the solder or by placing one end of thebody tube within the solder tube and one end over the solder tube andapplying energy to effect bonding. Where the tube to be repairedincludes a damaged section which requires replacement a graft materialwith solder applied at least at the ends can be joined at either end toa free end of the severed tube (FIG. 9).

Where the tissue repair is with respect to nerve tissue or other tissuetubes where the tube contents need to be protected from damage, it isespecially important that the weld should not be concentrated on theedges being joined as this can damage extruded tissue. Rather, the weldshould be transverse to the edge of the discontinuity.

The solders of the invention can be used, in conjunction with suitablepromoters of neuron growth, in tubular form, to provide guides for nerveregeneration. In this use the severed nerve ends are inserted into theends of the tube and welded in place.

The solders of the invention can also be used in tubular form with asealed end as a cap for the ends of severed nerves to assist patientswho experience discomfort, which can be extreme, where severed nervescannot be rejoined, for instance, in amputation stumps.

Where the tissue to be repaired is an essentially wide hollow body tube,the repair can comprise the insertion of a thin-walled hollow cylinderof bio-degradeable solder inside the tube under repair so that thecylinder spans the severed portions of the tube.

End-to-end repairs can also be performed by pulling one end of therepair site through the tube and folding back a cuff of tissue over thetube and then sleeving the other end over the cuff and effecting weldsto hold the tube and ends in place. It will be understood that in thisparticular method it is necessary for the energy source chosen to effectthe weld to propagate through the is overlying tissue.

Repairs of tubes in accordance with the invention can includeend-to-side as well as end-to-end tubular repairs.

End to side repairs can be performed by providing a tube with a flangeat one, end adapted to fit into a x-shaped incision in the side of thetube into which the end is to be inserted. The free tubular end of thesolder tube is attached to the end of the tube to be inserted into thex-shaped incision. The sides of the x-shaped incision are welded aroundthe circumference of the solder tube to seal the insertion site. Theend-to-side join can be at a variety of angles and thus the flangedportion of the tube can be provided at the appropriate angle for thejoin to be formed.

The repair methods of the invention may be utilised for joining adiversity of living tubular tissues including arteries, veins,lymphatics, microvessels, any of the body's tubes such as itsducts—pancreatic, liver, cystic, tear, prostatic, and the ureters,urethra, epididymis, vas, fallopian tubes, bowel, bronchi and othergastroenterological and respiratory and body and brain ducts and tubes.

The repair method of the invention can also be applied to the repair oforgans and their coverings such as liver, spleen, kidney, uterus,testicles, bladder, cystic, correal, brain and other capsules, coveringsand skin and appendages, as well as the various internal and peripheralnerves of the body, the spinal cord and its ramifications by use of atleast one appropriately shaped solder of the invention for the repairbeing made.

The present invention provides a new system of laser-solder-fusion, withor without control of the laser operation which we have demonstrated tobe suitable for joining together to produce usual function, in severedliving tubes in the rodent, namely arteries, veins, nerves and the vasdeferens and bowel. Not only are these severed tubular structures joinedwithout subsequent leakages but they function immediately after joining,those joins are at least eventually as strong and long lasting as ispossible with appropriate sutures, they are able to be joined in anexceptionally short time and in addition this is done without inflictingthe trauma occasioned by other methods. The system can be adapted co beused through equipment now and in the future developed for minimallyinvasive therapies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a solid protein cylinder of the invention measuring 2 mm inlength and 1.1 mm inner diameter and 1.3 mm outer diameter.

FIG. 2 shows a schema of an operative technique of the fourth aspect ofthe invention: (A) The solder is pushed over the proximal vessel end andthe vessel wall is pulled back. (B) Laser energy application at thedistal part of the solder. (C) The distal end of the vessel is gentlypulled over the entire length of the solder. (D) Laser energyapplication to the proximal part of the solder.

FIG. 3 shows the appearance of the laser welded micro-anastomosisimmediately after clamp release (A) and after 6 weeks (B).

FIG. 4 shows graphic representation of tensile strength of suture andlaser solder anastomoses of rat aortas as a function of time aftersurgery. (time is on logarithmic scale)

FIG. 5 shows a laser-welded anastomosis in longitudinal sectionimmediately after laser irradiation. (Masson's trichrome, (A) arrowindicates direction of blood flow. 5× magnification and (B) 50×magnification)

FIG. 6 shows the remains of solder in the vessel wall after 6 weeks.Note the normal appearance of the intima and media. Note the presence ofphagocytotic cells at the solder surface. (Toluldine Blue, magnification20×)

FIG. 7 shows scanning electron micrographs of the lumen of the laserwelded anastomosis 10 minutes after reestablishing perfusion(longitudinal section). (magnification×100).

FIG. 8 is a schematic cross section of an anastomosis of a blood vesselformed using the sleeve technique.

FIG. 9 shows a graft in side and cross sectional view formed using thesleeve technique of the fourth aspect of the invention at both ends ofthe graft.

FIG. 10 shows in schematic form, a join formed by placing a solder tubeof the invention inside a body tube. Solder strips may be usedexternally to strengthen the anastomosis.

BEST METHOD OF CARRYING OUT THE INVENTION

1. Preparation of Solder

Starting of Composition: protein 55–75% (w/w) water 45–25% (w/w) dye0.25% (w/w)The protein is bovine, rabbit, human, ovine or rat albumin. Suitableconcentrations for bovine serum albumin include about 55% and for humanand rabbit albumin include about 57% Indocyanine green is a suitabledye. Albumins can be obtained from Sigma-Aldrich Corporation. Suitablealbumin preparations include:Bovine albumin—A 2153 Fraction V powder (minimum 96%);Human albumin—A 1653 Fraction V powder (96–99% albumin);Rabbit albumin—A 0639 Fraction V powder;Sheep albumin—A 3264 Fraction V powder;Horse albumin—A 9888 Fraction V powder.ovine albumin.Indocyanine green dye can be obtained from Becton Dickinson MicrobiologySystems, Maryland 21030 USA.A particular formulation for human and rabbit albumin is as follows:

Starting of Composition: albumin 57.3% (w/w) water 42.45% (w/w) ICG dye0.25% (w/w)Construction:

1. the components (accurately measured) are mixed into a paste form toobtain optimum consistency for extrusion or pressing. For example, thewater and dye are first mixed by vortexing to form a consistent dyesolution which is then added to the protein followed by mixing to formthe paste. Mixing can be performed physically or mechanically and forsmall batches (<2 g total mass) was performed using a vortex mixer toprovide consistency. The solder was not allowed to dry at this stage asthis would cause the solder to become brittle and thus unsuitable forextrusion or pressing.

2. The paste can be extruded at this stage but as noted below a superiorproduct can be achieved by deferring final shaping.

3. The extruded paste was then allowed to dehydrate thus increasing theprotein concentration and allowing the solder to take a more rigid form.

4. The rigid solder was immersed in hot water at 80–90° C. (for example85° C. for bovine albumin) for approximately 1 minute to denature theprotein. Where the solder is prepared from human or rabbit albumin therelevant treatment is with steam at about 120° C. for 10 minutes (it isenvisaged that the temperature could be as low as 100° C. or up to 150°C.). This denaturation treatment causes the solder to bond within itselfand the solder becomes less soluble in water.

5. The solder at this stage is elastic and may be further cut intodesired shapes easily without inducing stress or fracture. Desiredshapes include sheets, tubes, partial tubes and rods. If cut to shapebefore step 4, the solder may fracture through the presence ofcrystalline structure if it is too dry or else it may deform if it istoo moist.

6. The solder is preferably dehydrated at this stage and gammairradiated or autoclaved for sterilisation and stored in a dry, sterileand light proof container.

A particular protocol that has been used successfully with the human orrabbit serum albumin formulation mentioned above is:

1. mix the protein preparation

2. extrude the preparation

3. allow the preparation to dry

4. autoclave the preparation at 120° C. for 10 minutes.

2. Method of Repair

The following repairs have been effected:

Rat aorta: 1.3 mm diameter cylinder used: 1.4 mm internal diameter 1.7mm external diameter,   2 mm length Rabbit femoral artery:   2 mmdiameter Cylinder used: 1.6 mm internal diameter 2.1 mm externaldiameter   2 mm length

Joining tubes can involve attaching at least one edge of thecircumference of a solder tube to the inside or outside of thecylindrical surface of a body tube. The join of the body tube can becompleted by placing both ends of the tube within the solder tube andapplying energy through the solder to bond the solder to the underlyingtissue or by placing both ends of the body tube over the solder tube andapplying energy through the overlying tissue to the solder or by placingone end of the body tube within the solder tube and one end over thesolder tube and applying energy to effect bonding. Where the body tubeto be repaired includes a damaged section which requires replacement agraft material with solder applied at least at the ends can be joined ateither end to a free end of the severed tube.

Where the tissue repair is with respect to nerve tissue or other tissuetubes where the tube contents need to be protected from damage, it isespecially important that the weld should not be concentrated on theedges being joined as this can damage extruded tissue. Rather, the weldshould be transverse to the edge of the discontinuity.

End to side repairs can be performed by providing a tube with a flangeat one end adapted to fit into a x-shaped incision in the side of thetube into which the end is to be inserted. The free tubular end of thesolder tube is attached to the end of the tube to be inserted into thex-shaped incision. The sides of the x-shaped incision are welded aroundthe circumference of the solder tube to seal the insertion site. Theend-to-side join can be at a variety of angles and thus the flangedportion of the tube can be provided at the appropriate angle for thejoin to be formed.

End-to-side repairs can also be performed by providing a partial soldertube with a flange adapted to fit over a longitudinal incision in theside of the body tube onto which the new tubular end is to be attached.The sides of the longitudinal incision are pulled through the solderflange, everted around the flange and welded to the outside of thesolder flange. The free end of the side branch is then pulled over thepreviously welded body tube and flange and welded to the main body ofthe partial solder tube. The main body of the partial solder tube isthen welded to the outside of the main body tube. The end-to-side joincan be at a number of angles and thus the flanged portion of the tubecan be provided at the appropriate angle.

Repairs of non-tubular tissues are effected by using at least oneappropriately shaped solder of the invention together with an energysource to effect bonding between solder and tissue.

3. Description of Sleeve Method

The proximal artery (tube) is pulled through a tube of solder and turnedback on itself a short distance using purpose built forceps, which haveends adapted to provide a surface which functions to maintain the tubeend in open form, such as the forceps illustrated in FIG. 2. Theoverlapping turned back artery is lasered to an observable slight changein colour and specific temperature, which denatures the protein andcauses it to adhere to the vessel wall on both or at least one side in acircle around the proposed join area. The distal artery (or tube) isslightly stretched and manipulated gently over the already lasered areaand beyond to the as yet unlasered solder tube of equal lasing area.This area is then lasered in the same way and causes that circularportion of the artery to be lasered to the cylinder. That completes thejoin.

EXAMPLE 1

A total of 90 rats were divided into two groups randomly. In group onethe anastomoses were performed using conventional microsuturingtechnique, while in group two the anastomoses were performed using ournew laser welding technique. In addition, each of the two groups weredivided into 5 subgroups and evaluated at different followup periods (10min, 1 hour, 1 day, 1 week and 6 weeks). At these intervals theanastomoses were evaluated for patency and strength (tensile strengthmeasurement) anastomoses in each subgroup were processed for light andelectron microscopy.

All anastomoses were found to be patent. The mean clamp time of theanastomoses performed with conventional suturing was 20.6 minutescompared to 7.2 minutes for the laser activated welded anastomoses(p<0.001). The strain measurements showed a stronger mechanical bond ofthe sutured anastomoses in the initial phase. However, at 6 weeks thetensile strength of the laser welded anastomoses was higher compared tothe conventional suture technique. Histologic evaluations revealed anear complete resorption of the solder after six weeks. The junctionsite of the vessel ends could not be determined on the luminal side ofthe artery.

In conclusion, a resorbable protein used as a solder, activated by adiode laser, can provide a reliable, safe and rapid arterialanastomosis, which could be performed by any microsurgeon faster thanconventional suturing after a short learning curve.

Simplifying vascular anastomoses in surgery and in particular in smalldiameter vessels has been an important topic in the past. A recentpublication reviewed the technical developments in this field since thestart of this century [1]. Minimising foreign body reaction at theanastomotic site has been an important issue, and a variety of authorshave described the negative impact of suture materials, staples andclips on vessel wall compliance and active force production [2–6]. Theuse of laser welding techniques for vascular anastomosis has first beenreported by Jain in 1979 [7, 8]. Different types of lasers hare beenused [9–12] in order to minimise the potential negative impact ontissues. Most reported techniques require at least three permanent staysutures and therefore laser welding was used only to seal the vessel andnot to mechanically hold the vessel ends together. The use of lasers toweld tissue relies on the efficient deposition of heat due to the lightabsorbed by the tissue. The laser wavelengths that have been used thuscorrespond to strong absorption bands of water, hemoglobin or othertissue chromatophores. The introduction of dyes such as indocyaninegreen [13, 14] or fluorescein isothiocyanate [15] enhances the deliveryof the laser energy precisely to the target tissues. In addition, theapplication of laser activated protein solders has been shown tostrengthen laser welds in tissues such as nerves [16–18]. Our studypresents a sutureless, quick and reliable technique to successfullyanastomose small diameter arteries, avoiding vessel wall fibrosis byeliminating any permanent implanted devices. We combined an anastomotictechnique reported by Payr in 1900 [19] with the use of a fullybiodegradeable, diode-laser-activated protein tube to weld smalldiameter arteries.

Materials and Methods

A total of 90 young adult male Wistar rats (outbred) weighing 450 to 550g were used in this study. Consent and approval for this investigationwere obtained from our Institution's Animal Ethical Review Committee.All surgical procedures were performed under general anaesthesia with ahalothane/oxygen mix (4% halothane at 4 L/min oxygen for inducing and 2%halothane at 2 L/min oxygen for maintaining anaesthesia). Clean, but notaseptic conditions were maintained during the surgical procedures, whichwere performed using a Zeiss OPMI 7 operating microscope. A midlinelaparotomy was performed and the infrarenal aorta exposed, incising theperitoneum, freeing the tissues and ligating lumbar and ileolumbarvessels if necessary. A double microvascular clamp (Edward Weck Inc.micro vessel approximator 1.5 mm×8.0 mm blades, 19 mm bar) was appliedto the aorta, which was severed with straight microscissors. Afterflushing the two stumps with saline, connective tissue in excess wasremoved, but leaving the adventitia intact. In 45 animals theanastomoses were carried out by conventional microsuturing (9/0 Nylonwith a 140 u needle, 10 to 12 interrupted sutures) and in the remaining45 animals the anastomoses were performed by laser welding. The clasptime of all procedures was recorded for later statistical analysis withthe students t-test. No local or systemic anticoagulant drugs were used,nor were the animals given antibiotics post-operatively.

Laser Welding

A GaAlAs laser diode with a nominal power of 250 mW and wavelength of805 nm (Spectra Diode Labs Inc., San Jose Calif.) was used. The laserradiation was coupled into a 100 um diameter core, numerical aperture(NA 0.28) optical fiber, which was held by hand in a fiber chuck. Thediode current and temperature were controlled by a SDL-800 diode driver.The diode was operated by a foot switch and was set at 90 mW duringsurgery, with a spot size at the tissue of 200 um diameter,corresponding to a maximum irradiance of 286 W/cm² at the tissuesurface. The laser power was measured with a Scientech power meter(Boulder Inc., CO USA). The total irradiation time for each circularweld was 10 sec approximately.

The solder used in this study was a mixture of water, concentratedbovine serum albumin and indocyanine green (ICG) dye (Becton Dickinson,Maryland USA). ICG has a maximum absorption coefficient at a wavelengthof 805 nm of 2×10⁵M⁻¹cm⁻¹. ICG binds preferentially with serum proteinssuch as albumin [20] ensuring that the heat is efficiently transferredto denature the protein solder. A high protein concentration mixture(55.40% albumin: 44.33% water: 0.27% ICG by weight starting material)was obtained by vigorous stirring of the components. The mixture wasformed into tubes suited to the dimensions of a rat aorta. The soldertubes were predenatured to make them more flexible and chemically stable(FIG. 1).

The solid protein tube was then used in a way similar to that describedby Payr in 1900 when using absorbable magnesium rings [19], (FIG. 2).The proximal vessel was passed through the cylinder, everted over theedge for a length of 1 mm and then welded to the protein cylinder bymeans of laser energy, further denaturating the protein contained in thesolder (FIGS. 2A, B). Laser energy was delivered by an optical hand-heldfiber for a period of time according to the tissue reaction visiblethrough the operating microscope (approximately 10 sec/circumference).When the slightest retraction of the tissue was noted the laser spot wasmoved to adjacent tissue until the total circumference of the vessel waswelded onto the protein cylinder. The two branches of the double clampwere then approximated and the distal vessel was gently pulled over theentire protein cylinder (FIG. 2C). Laser energy was then applied tocreate a bond between the distal end of the artery and the most proximalpart of the solder (FIG. 2D).

Immediately after removing the clamps the anastomoses were examined toassess patency by the milking test. Each group was then divided into 5subgroups to be reevaluated at different intervals (10 minutes, 1 hour,1 day, 1 week, 6 weeks) with 9 animals per subgroup. At the chosen timeall anastomoses were re-exposed and patency was checked with the milkingtest. In 6 animals per subgroup the anastomotic sites together with 5 mmof vessel proximally and distally were removed and subjected to tensilestrength measurements. These were performed by attaching one end of thevessel to a calibrated force transducer (FT30C, Grass Instruments,Quincy, Mass.) and the other end to a screw driven translator [18]. In 3animals per subgroup the vessels were clamped, flushed with saline andfixative (5% glutaraldehyde buffered to pH 7.4) and finally removed forhistology. Staining for light microscopy was done with Masson'strichrome to clearly differentiate native protein from denatured proteinand with Toluidine Blue. Scanning electron microscopy was used to studythe inner surfaces of the anastomoses.

Results

All animals survived the surgical procedure and all anastomoses werepatent at the time of re-exploration. At 6 weeks there were no aneurysmsat the site of the sutured or laser welded anastamoses (FIG. 3).

The mean clamp time of the sutured anastamoses was 20.6 minutes (SD2.82, SEM 0.52) which was significantly longer than the mean clamp timeof the laser welded anastomoses, 7.2 minutes. (SD 2.26, SEM 0.41),(p<0.001; students t-test).

Tensile strength measurements revealed that the sutured anastomoses werestronger (under stress) when compared to the laser-welded anastomoses inthe short term (134.6 gm and 45.3 gm respectively). However, at 6 weeksthe tensile strength for the laser welded anastomoses was slightlyhigher in comparison to the sutured anastomoses (134.2 gm and 103.9 gmrespectively, p=0.005, student's t-test) (FIG. 4) The suturedanastomoses, when subjected to traction, ruptured at the junction leveltearing a small cuff off the vessel wall, while the laser-welded vesselsdetached at the distal portion of the bond, probably the weakest pointof the anastomosis.

Light microscopy evaluation after staining of the anastomotic siteimmediately after laser application with Masson's trichrome revealeddenaturated protein in the layer directly adjacent to the solder, but nochanges could be observed in the media of the artery (FIG. 5). After 6weeks the solder was almost completely resorbed and the intimal layercould be observed in continuity. Healing occurred with proliferation ofmyofibroblasts and the site of the anastomosis could not be detectedfrom the lumen of the artery (FIG. 6). However, some fibrotic reactioncould be seen on the adventitia as shown in FIG. 3 b, but again themedia of the artery did not reveal any changes. Scanning electronmicroscopy of the anastomotic site after perfusion was reestablished for10 minutes showed some red blood cell deposition at the site of theanastomosis, but this did not have any impact on patency (FIG. 7).

Discussion

Since Jain's first report on the successful use of laser energy for therepair of blood vessels [7], there have been numerous attempts todevelop a technique for sutureless anastomosis of blood vesselsemploying laser welding. The advantages of laser welding have been shownto be due to a perfect seal of the junction with no leakage and lessforeign body reaction due to less suture material. However, the need forstay sutures to maintain vessel end approximation has led to the termlaser-assisted anastomosis [21–25], where the laser is used to seal ananastomosis after three to five stay sutures have been previouslyinserted. On the other hand, true sutureless anastomoses have beenperformed by using an intraluminal stent to ensure intimal alignment[26–28]. These stents have mostly been designed to be intraluminallyabsorbed and therefore may potentially lead to arterial embolism and/orthrombosis. A previously reported technique to repair tubular structures[18] employs protein solder bands containing indocyanine green dye,which were designed to absorb the laser energy and therefore heat waslocalized at the protein solder and the immediate surrounding tissue.

Changes in the tissue due to heating caused by the laser energy wereobserved only in the tissue layer in immediate contact with the solder.In order to get optimal intimal alignment, which is crucial forsuccessful microvascular anastomosis, the protein solder was extrudedinto a tube with corresponding diameters to the vessel to be repaired.The optimal intimal alignment was accomplished by employing a techniqueintroduced by Payr at the turn of the century [19]. This technique wasfurther developed by Landon [29] eliminating the need for ligatures tosecure the vessel onto the ring and by Carter [30] for coronary arterysurgery using a polyethylene ring. Haller [31] reported a 92% patencyrate in the anastomosis of 4-mm diameter vessels using Payr's techniquewith tantalum rings. This technique prevents the blood coming in contactwith the protein solder which eliminates the risk that the coagulationcascade is activated and leads to smooth intimal alignment. The laserwelding technique causes the tissue to bond to the protein solder tubeby means of protein denaturation in the tissue and the solder.

In earlier studies the actual mechanism of the bond created by laserwelding has been identified as the possible homogenisation of theadventitia as well as coagulation necrosis of smooth muscle cells,however the elastic lamellae were unaltered [32]. In a direct laserwelding study, protein denaturation of the collagen fibers was observedwith electron microscopy, with a slight interruption of the intima andsubsequent re-endothelialization within 10 days [33]. Dehydration of thetriple helix molecular structure of collagen present in the arterialwall breaking Van der Waal's bonds, which subsequently re-form to othercollagen molecules, was reported as a possible bonding mechanism [34].Our technique confines the laser-induced changes in the artery wall tothe layer directly in contact with the protein solder, thus minimizingany weakening of the vessel wall. In particular neither the proximal nordistal vessels' tunica media were altered by the laser energy as shownby histologic evaluation. It may then be suggested, that by thistechnique the arterial wall is only minimally altered and does not loseits mechanical properties. After healing of the anastomotic site thetensile strength measurements revealed better results for thelaser-welded anastomoses compared to the sutured anastomoses, whichcould be a result of the fibrous reaction to the suture material in thetunica media.

REFERENCES

-   1) Werker P M N, Kon M. Review of facilitated approaches to vascular    anastamosis surgery. Ann. Thorac. Surg. 63:S122, 1997.-   2) Serure A, Withers E H, Thomson S, Morris J. Comparison of carbon    dioxide laser-assisted microvascular anastomosis and conventional    microvascular sutured anastomosis. Surg. Forum. 34:634, 1983.-   3) Lidman D, Daniels R K. The normal healing process of    microvascular anastomoses. Scan. J. Plast. Surg. 15:103, 1981.-   4) Servant J, Ikuta Y, Harada Y. A scanning electron microscope    study of microvascular anastomoses Plast. Reconstr. Surg. 57:329,    1976.-   5) Acland R D, Trachtenberg L The histopathology of small arteries    following experimental microvascular anastomosis. Plast Reconstr.    Surg. 59:868, 1977-   6) Dalsing M C, Packer S C, Kueppers P, Griffith S L, Davis T B.    Laser and suture anastomosis: Passive compliance and active force    production. Lasers Surg. Med. 12:190, 1992.-   7) Jain K K, Gorisch W. Repair of small blood vessels with the    Neodymium-Yag laser. A preliminary report Surgery 51:684, 1979.-   8) Jain K K, Gorisch W. Microvascular repair with Neodymium-Yag    laser. Acta Neurochir. (Wien) Suppl. 28:260, 1979.-   9) White R A, Abergel R P, Lyons R, Klein S R, Kopchok G, Dweyer R    M, Uitto J. Biological effects of laser welding on vascular healing.    Lasers Surg. Med. 6:137, 1986.-   10) Kopchok G E, White R A, White G H, Fujitani R, Vlasak J,    Dykhovsky L, Grundfest W S. CO₂ and argon laser vascular welding.    Acute histologic and thermodynamic comparison Lasers Surg. Med.    8:584, 1988.-   11) Nakata S. Campbell C D, Pick R, Replogle R L. End-to-side and    end-to-end vascular anastomoses with a carbon dioxide laser J.    Thorac. Cardiovasc. Surg. 98:57, 1989.-   12) Lewis W J. Uribe A. Contact diode laser Microvascular    anastomosis. Laryngosc. 103:850, 1993.-   13) Reali T M, Gelli R, Gianotti V, Clori F, Pratesi R, Pini R.    Experimental diode laser-assisted microvascular anastomosis. J.    Reconstr. Microsurg 3:203, 1993.-   14) Oz, M C, Johnson J P, Paranagi S, Chuck R S, Marboe C C, Bass L    S, Nowygrod R, Treat M R. Tissue soldering by use of indocyanine    green dye-enhanced fibrinogen with near infrared diode laser. J.    Vasc. Surg. 11:718, 1990.-   15) Chuck R S, OZ M C, Delohery T M, Johnson J P, Bass L S, Nowygrod    R, Treat M R. Dye-enhanced laser tissue welding. Laser Surg. Med.    9:471, 1989.-   16) Bass L S, Moazami N, Avellino A, Trosaborg W, Treat M R.    Feasibility studies for laser solder neuro-rhaphy. Proc SPIE    2128:472, 1994.-   17) Menovsky T, Beek J F, van Gemert M J C. CO₂ laser nerve welding    optimal laser parameter and the use of solders in vitro. Microsurg.    15, 44, 1994.-   18) Lauto A, Trickett R, Malik R, Dawes J M, Owen E R.    Laser-activated solid protein bands for peripheral nerve repair. An    in vivo study. Laser Surg. Med. 21:13 4, 1997.-   19) Payr E. Beitraege zur Technique der Blutgefaessund Nervennaht    nebst Mittellungen ueber die Verwendung eines resorblerbaren    Metalles in der Chirurgle. Arch. Klin. Chir. 62:67, 1900.-   20) Sauda K, Imasaka T, Ishibashi N. Determination of protein in    human scrum by high performance liquid chromatography. Analytical    Chemistry 58: 2649, 1986.-   21) McCarthy WJ, LoCicero J, Hartz R S, Yao J S T. Patency of    laser-assisted anastomoses in small vessels: Oneyear follow-up.    Surgery 102.319, 1987.-   22) Okada M, Shimizu K, Ikuta Horii H, Nakamura K. An alternative    method of vascular anastomosis by laser: experimental and clinical    study. Laser Surg. Med. 7:240, 1987.-   23) Abrahamson D L, Shaw W W, Kamat B R, Harper A, Rosenberg C R.    Laser-assisted venous, anastomosis: A comparison study. J. Reconstr.    Microsurg. 7:199, 1991.-   24) Kiyoshige Y, Tsuchida H, Hamasaki M, Takayanagi M, Watanabe Y.    CO₂ laser-assisted microvascular anastamosis: Biomechanical studies    and clinical applications. J. Reconstr. Microsurg. 7:225, 1991.-   25) Tang J. Godlewski G, Rouy S, Dauzat M, Juan J M, Chambettaz F,    Salathe R. Microarterial anastomosis using a noncontact diode laser    verses a control study. Users Surg. Med. 14:229, 1994.-   26) Jain K K. Sutureless microvascular anastomosis using a    Neodymium-YAG laser. J. Microsurg. 1:436, 1980.-   27) Niijima K H, Yonekawa Y, Handa H, Taki W. Nonsuture microvasular    anastomosis using an Nd-YAG laser and a water-soluble polyvinyl    alcohol splint. J. Neurosurg. 67:579, 1987.-   28) Bass L S, Treat M R, Dzakonski C, Trokel S L. Sutureless    microvasular anastomosis using the THC:YAG laser. A preliminary    report. Microsurg. 10: 189, 1989.-   29) Landon L H. A simplified method of direct blood transfusion with    self retaining tubes. JAMA 61:490, 1913.-   30) Carter E L, Roth Ej. Direct non-suture coronary anastomosis in    the dog. Ann. Surg. 148:212, 1958.-   31) Haller J D, Kripke D C, Rosenak S S, Roberts D R, Rohman M.    Long-term results of small vessel anastomoses with a ring technique.    Ann. Surg. 161:67, 1965.-   32) Schober R, Ulrich F, Sander T, Duerselen H, Hessel S.    Laser-induced alteration of collagen substructure allows    microsurgical tissue welding. Science 232:1421, 1986.-   33) Godlewski G, Rouy S, Dauzat M. Ultrastructural study of arterial    wall repair after argon laser micro-anastomosis. Lasers Surg. Med.    7:258, 1987.-   34) Fenner J, Martin W, Moseley H, Wheatley Dj. Shear strength of    tissue bonds as a function of bonding temperature: a proposed    mechanism for laser-assisted tissue welding. Lasers Med Science    7:39, 1992.

1. A biomolecular solder made by a method comprising (b) providing acomposition comprising a proteinaceous substance in a solvent; and (b)pre-denaturing the proteinaceous substance before placing thecomposition in situ by at least partially denaturing the proteinaceoussubstance while moist with the solvent such that at least a portion ofthe proteinaceous substance bonds together.
 2. A solder according toclaim 1 wherein the proteinaceous substance comprises a protein.
 3. Asolder according to claim 2 wherein the protein comprises an albumin, acollagen an elastin, a fibrinogen, or any combination thereof.
 4. Asolder according to claim 1, further comprising a dye.
 5. A solderaccording to claim 4 wherein the dye comprises an indocyanine green, amethylene blue or a fluorescein isothiocyanate or any combinationthereof.
 6. A solder according to claim 1, further comprising anadjuvant.
 7. A solder according to claim 1 further comprising a growthfactor, a sodium hyaluronate, a hormone and an anti-coagulant.
 8. Asolder according to claim 1 further comprising a material for improvingthe strength of the solder.
 9. A solder according to claim 8 wherein thematerial comprises a polytetrafluoroethylene fibre or a ceramic fibre.10. A kit comprising a biomolecular solder according to claim
 1. 11. Amethod of preparing a biomolecular solder ex vivo, the methodcomprising: (a) providing a composition comprising a proteinaceoussubstance and a solvent; (b) shaping the composition into a desiredshape, wherein the composition is shaped before, during or after thepre-denaturing of step (c), or a combination thereof; and (c)pre-denaturing the proteinaceous substance before placing thecomposition in situ by at least partially denaturing the proteinaceoussubstance while the composition is moist such that at least a portion ofthe proteinaceous substance bonds together, thereby preparing abiomolecular solder.
 12. A method according to claim 11 wherein theproteinaceous substance is pre-denatured by exposing the solder to anenergy for a time period that is sufficient to allow the energy to atleast partially denature the proteinaceous substance.
 13. A methodaccording to claim 12 wherein the energy comprises a thermal energy. 14.A method according to claim 13 wherein the proteinaceous substance ispre-denatured by heating the solder at a temperature of greater than 40°C. for a time period of about 30 seconds or longer.
 15. A methodaccording to claim 14 wherein in the pre-denaturing step the solder isheated in a hot liquid bath or in pressurized steam.
 16. A methodaccording to claim 11 wherein the proteinaceous substance ispre-denatured by exposure to a denaturing agent for a time period thatis sufficient to allow the denaturing agent to homogenously andcompletely denature the proteinaceous substance.
 17. A method accordingto claim 11 wherein the biomolecular solder further comprises a dye. 18.A method according to claim 17 wherein the dye is in an amount between0.1 to 2.5% w/w of the solder.
 19. A method according to claim 17wherein the dye is mixed with the solvent, prior to mixing the solventwith the proteinaceous substance.
 20. A method according to claim 11wherein the pre-denaturing step further comprises drying thecomposition, wherein a majority of the solvent is removed from thecomposition during the drying of the composition.
 21. The method ofclaim 11 wherein in the pre-denaturing step the composition is appliedto a support structure before the proteinaceous substance ispre-denatured.
 22. The method of claim 21 wherein the support structureis a mesh, a stiffener or a graft material.
 23. The method of claim 11further comprising the step of sterilizing the biomolecular solderfollowing the pre-denaturing of the proteinaceous substance.
 24. Amethod of welding or joining a biological tissue together, the methodcomprising: (a) applying a biomolecular solder according to claim 1 tothe biological tissue to be welded or joined together; and (b) exposingthe biomolecular solder to an energy for a time sufficient to cause thesolder to weld or join the biological tissue together.
 25. The method ofclaim 24 wherein the pre-denatured solder is moistened beforeapplication to the biological tissue.
 26. The biomolecular solder ofclaim 1 wherein the proteinaceous substance is denatured ex vivo suchthat it is essentially insoluble in the physiological fluid at bodytemperature.
 27. The biomolecular solder of claim 1 wherein thepre-denatured solder has been shaped from a composition comprising theproteinaceous substance in an amount of at least 40% w/w of thecomposition.
 28. The biomolecular solder of claim 1 wherein theproteinaceous substance comprises at least one substance selected fromthe group consisting of a protein, a polypeptide, a mixture of proteins,a biodegradable protein, a fibrous material, a synthetic polypeptide andany combination thereof.
 29. The method of claim 11 further comprisingdrying the pre-denatured solder.
 30. The method of claim 11 wherein thepre-denatured solder, shaped into the predetermined shape, comprises theproteinaceous substance in an amount of at least 40% w/w or greater ofthe solder.
 31. The method of claim 11, wherein the solder initiallycomprises a proteinaceous substance in an amount in the range from 50%w/w to 80% w/w of the solder.
 32. The method of claim 30 or 31 whereinthe solder initially comprises a solvent in an amount up to 60% w/w ofthe solder.
 33. The method of claim 11 wherein the pre-denaturing stepcomprises heating the solder at a temperature in a range from betweenabout 75° C. to 100° C.
 34. The method of claim 33 wherein thepre-denaturing step comprises heating the solder at a temperature in arange from between about 100° C. to 150° C.
 35. The method of claim 16wherein in the pre-denaturing step the denaturing agent comprises achemical.
 36. The method of claim 11, wherein the proteinaceoussubstance comprises at least one substance selected from the groupconsisting of a protein, a polypeptide, a mixture of proteins, abiodegradable protein, a fibrous material, a synthetic polypeptide andany combination thereof.
 37. The method of to claim 36 wherein theproteinaceous substance comprises at least one substance selected fromthe group consisting of human albumin, bovine albumin, horse albumin,ovine albumin, rabbit albumin, rat albumin, and a combination thereof.38. The method of claim 36, wherein the proteinaceous substancecomprises at least one protein selected from the group consisting of analbumin, an elastin, a collagen and a fibrinogen.
 39. The method ofclaim 25 wherein the moistening of the pre-denatured solder increasesflexibility of the solder.
 40. The biomolecular solder of claim 1,wherein the solvent comprises an aqueous solvent.
 41. The biomolecularsolder of claim 40, wherein the aqueous solvent comprises water orsaline.
 42. The method of claim 11, wherein the solvent comprises anaqueous solvent.
 43. The method of claim 42, wherein the aqueous solventcomprises water or saline.
 44. The method of claim 11, whereindenaturing the protein in situ in step (e) comprises denaturing theproteinaceous substance by exposing the solder to a laser energy. 45.The method of claim 44, wherein the laser is a diode laser.
 46. Themethod of claim 24, wherein the biological tissue is welded together toeffect a repair.
 47. The biomolecular solder of claim 1, whereindenaturing the protein in situ in step (e) comprises denaturing all ofthe proteinaceous substance.
 48. The biomolecular solder of claim 1,wherein denaturing the protein in situ in step (e) comprises denaturinga portion of the proteinaceous substance.
 49. The method of claim 11,wherein denaturing the protein in situ in step (e) comprises denaturingall of the proteinaceous substance.
 50. The method of claim 11, whereindenaturing the protein in situ in step (e) comprises denaturing aportion of the proteinaceous substance.
 51. The biomolecular solder ofclaim 1, wherein the method of making the solder further comprisessterilizing the biomolecular solder before the step (d) placing of thepre-denatured solder in situ.
 52. The biomolecular solder of claim 1,wherein the pre-denatured proteinaceous substance is shaped into asheet, a tube, a partial tube, a strip, a patch, a hollow tube with aflanged end or a rod before the step (d) placing of the pre-denaturedsolder in situ, after the step (d) placing the pre-denatured solder insitu, or a combination thereof.
 53. The method of claim 11, the desiredshape comprises a sheet, a tube, a partial tube, a strip, a patch, ahollow tube with a flanged end or a rod before the step (d) placing ofthe pre-denatured solder in situ, after the step (d) placing thepre-denatured solder in situ, or a combination thereof.
 54. Abiomolecular solder comprising a protein comprising an albumin, anelastin, a collagen, a fibrinogen or a combination thereof, wherein thebiomolecular solder is made by the method of claim 1, and thepre-denatured solder has been at least partially denatured while moistsuch that the protein bonds together and, when shaped, the shape of thesolder is thereby essentially maintained and the solubility of theprotein is reduced in a physiological fluid at body temperature.
 55. Thebiomolecular solder of claim 54, wherein the solder is shaped beforepre-denaturing.
 56. The biomolecular solder of claim 54, wherein thesolder is shaped after pre-denaturing.
 57. The biomolecular solder ofclaim 54, wherein the protein comprises a bovine, rabbit, ovine, rat orhorse serum albumin.
 58. The biomolecular solder of claim 54, whereinthe protein comprises a human albumin, a human elastin, a humanfibrinogen, a human collagen or any combination thereof.
 59. Thebiomolecular solder of claim 54, further comprising a dye for improvingenergy deposition into the solder when the solder is exposed to energy.60. The biomolecular solder of claim 54, wherein the proteinaceoussubstance has been at least partially denatured while moist with asolvent.
 61. The biomolecular solder of claim 60, wherein the solventcomprises an aqueous solvent.
 62. The biomolecular solder of claim 61,wherein the aqueous solvent comprises water or saline.
 63. Abiomolecular solder made by a method comprising: (a) providing acomposition comprising a protein in a solvent; (b) pre-denaturing theprotein before placing the composition in situ by at least partiallydenaturing the protein while moist with the solvent such that at least aportion of the protein bonds together; and, (c) shaping thepre-denatured protein, wherein the solder is shaped before, during orafter the pre-denaturing of step (b), or a combination thereof.
 64. Thebiomolecular solder of claim 63, further comprising steps (d) placingthe pre-denatured solder in situ, and (e) further denaturing the proteinin situ such that the final shape of the in situ-denatured solder isessentially maintained and the solubility of the protein is reduced in aphysiological fluid at body temperature.
 65. The biomolecular solder ofclaim 63, wherein the protein comprises albumin.
 66. The biomolecularsolder of claim 65, wherein the albumin comprises human albumin, bovinealbumin, ovine albumin, horse albumin, rat albumin or a mixture thereof.67. The biomolecular solder of claim 63, wherein the protein comprisescollagen, elastin, fibrinogen or a combination thereof.
 68. Thebiomolecular solder of claim 63, wherein pre-denaturing the proteinbefore placing the composition in situ comprises the step of steamheating or immersion into hot water.
 69. The biomolecular solder ofclaim 68, wherein the steam heating step comprises use of a temperatureof between about 100° C. and 150° C.
 70. The biomolecular solder ofclaim 63, wherein pre-denaturing the protein before placing thecomposition in situ comprises use of light, heat, radiation, ultrasoundor chemicals.
 71. The biomolecular solder of claim 63, wherein the stepof denaturing the protein in situ comprises exposing the solder tolight, heat, radiation, ultrasound or chemicals.
 72. The biomolecularsolder of claim 63, wherein the step of denaturing the protein in situcomprises exposing the solder to a laser energy.
 73. The biomolecularsolder of claim 72, wherein the laser energy that denatures the proteinin situ comprises a power of about 90 mW and a wavelength of about 805nm.
 74. The biomolecular solder of claim 72, wherein the laser energythat denatures the protein in situ comprises a spot size at the solderof about 200 μm.
 75. The biomolecular solder of claim 63, whereinfurther comprising a dye.
 76. The biomolecular solder of claim 75,wherein the dye comprises an indocyanine green, a methylene blue or afluorescein isothiocyanate.
 77. The method of claim 24, wherein thebiological tissue is a human or an animal tissue.
 78. The method ofclaim 24, wherein a blood vessel, a nerve, a pancreatic duct, a livervessel or duct, a cystic duct, a tear duct, prostatic duct, a ureter,urethra, an epididymis, a vas deferens, a fallopian tube, a bowel, abronchi, a gastroenterological tube or duct, a respiratory tube or ductor a brain vessel, tube or duct are welded together.
 79. A solderaccording to claim 1, wherein in step (b) the proteinaceous substance isfully denatured.
 80. The biomolecular solder of claim 1 wherein thecomposition comprises a proteinaceous substance in a concentration in arange of between about 40% w/w and 80% w/w, or between about 45% w/w and75% w/w, of the composition.
 81. A biomolecular solder made by a methodcomprising (a) providing a composition comprising a proteinaceoussubstance in a solvent; (b) pre-denaturing the proteinaceous substancebefore placing the composition in situ by at least partially denaturingthe proteinaceous substance while moist with the solvent such that atleast a portion of the proteinaceous substance bonds together and thesolubility of the proteinaceous substance is reduced in a physiologicalfluid at body temperature; and, (c) shaping the proteinaceous substance,wherein the solder is shaped before, during or after the denaturing ofstep (b), or a combination thereof, and, when shaped, the final shape ofthe solder is essentially maintained.
 82. A sterile biomolecular soldermade by a method comprising (a) providing a composition comprising aproteinaceous substance in a solvent; (b) pre-denaturing theproteinaceous substance ex vivo by at least partially denaturing theproteinaceous substance while moist with the solvent such that at leasta portion of the proteinaceous substance bonds together; and, (c)sterilizing the pre-denatured solder.
 83. A biomolecular soldercomposition comprising a shaped proteinaceous substance and a solvent,wherein the proteinaceous substance is at least partially denatured exvivo while moist with the solvent such that at least a portion of theproteinaceous substance bonds together.
 84. The composition of claim 83,wherein the proteinaceous substance is fully denatured.
 85. Thecomposition of claim 83, wherein the protein comprises an albumin, acollagen, an elastin, a fibrinogen, or any combination thereof.
 86. Asterile shaped biomolecular solder comprising an at least partiallycross-linked proteinaceous substance and a solvent, wherein theproteinaceous substance is at least partially cross-linked while moistwith the solvent such that at least a portion of the proteinaceoussubstance bonds together.
 87. A biomolecular solder comprising an atleast partially cross-linked protein and a solvent, wherein the proteincomprises an albumin, a collagen, an elastin, a fibrinogen, or anycombination thereof, and is at least partially cross-linked while moistwith the solvent.
 88. A kit comprising the sterile biomolecular solderof claim
 82. 89. A kit comprising the sterile shaped biomolecular solderof claim
 86. 90. A kit comprising the biomolecular solder of claim 87.91. A kit comprising the sterile biomolecular solder of claim 87, thesterile shaped biomolecular solder of claim 86 or the biomolecularsolder of claim 87, and instructions for using the solder as set forthin claim 24.